An increasing number of compounds able to potentiate the activity of mutants of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have been identified by high throughput screening or by individual search of derivatives of known active compounds. Several lines of evidence suggest that most CFTR potentiators act through the same mechanism, probably by binding to the nucleotide binding domains to promote the activity of the protein and then, with lower affinity, to an inhibitory site. With the aim of identifying the activating binding site, we recently modeled the nucleotide binding domain dimer and predicted a common binding site for potentiators in its interface. To validate this model experimentally, we mutated some of the residues involved in the putative binding site, i.e. Arg553, Ala 554, and Val1293. The activity ofCFTR potentiators was measured as apical membrane currents on polarized cells stably expressing wild type or mutated proteins. CFTR activity was elicited by application of a membrane-permeable cAMP analogue followed by increasing concentrations of potentiators. We found that all three mutants responded to cAMP, although the affinity of R553Q was higher than that of wild type CFTR. In R553Q and V1293G mutants, the dissociation constant of potentiators for the activating site was increased, whereas the dissociation constant for the inhibitory site was reduced. Our results show that the mutated residues are part of the activating binding site for potentiators, as suggested by the molecular model. In addition, these results suggest that the activating and inhibitory sites are not independent of each other.
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